Extracellular ATP induces unconventional release of glyceraldehyde-3-phosphate dehydrogenase from microglial cells.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key glycolytic enzyme that is predominantly localized in the cytoplasm. However, recent studies have suggested that GAPDH is released by various cells and that extracellular GAPDH is involved in the regulation of neuritogenesis in neuronal cells. It has also been reported that GAPDH is expressed on the surfaces of macrophages and functions as a transferrin receptor. However, since GAPDH is a leaderless protein the mechanisms by which it reaches the extracellular environment remain unclear. Here, we examined the role of P2X7 receptor (P2X7R), an ATP-gated cation channel, in the unconventional release of GAPDH from microglial cells, the resident macrophages in the brain. The activation of P2X7R by ATP triggered GAPDH release from lipopolysaccharide (LPS)-primed microglial cells. ATP-induced microvesicle formation, exosome release, and K(+) efflux followed by caspase-1 activation are likely involved in the GAPDH release, but ATP-induced dilatation of membrane pores and lysosome exocytosis are not. It was also demonstrated that exogenous GAPDH facilitated LPS-induced phosphorylation of p38 MAP kinase in microglial cells. These findings suggest that P2X7R plays an important role in the unconventional release of GAPDH from microglial cells, and the GAPDH released into the extracellular space might be involved in the regulation of the neuroinflammatory response in the brain.

Neuroanatomical distribution of disease-associated prion protein in experimental bovine spongiform encephalopathy in cattle after intracerebral inoculation.

The pathologic disease-associated prion protein (PrP(Sc)) has been shown to be expressed in the central nervous system of Holstein cattle inoculated intracerebrally with 3 sources of classical bovine spongiform encephalopathy (BSE) isolates. Several regions of the brain and spinal cord were analyzed for PrP(Sc) expression by immunohistochemical and Western blotting analyses. Animals euthanized at 10 months post-inoculation (mpi) showed PrP(Sc) deposits in the brainstem and thalamus, but no vacuolation; this suggested that the BSE agent might exhibit area-dependent tropism in the brain. At 16 and 18 mpi, a small amount of vacuolation was detected in the brainstem and thalamus, but not in the cerebral cortices. At 20 to 24 mpi, when clinical symptoms were apparent, heavy PrP(Sc) deposits were evident throughout the brain and spinal cord. The mean time to the appearance of clinical symptoms was 19.7 mpi, and the mean survival time was 22.7 mpi. These findings show that PrP(Sc) accumulation was detected approximately 10 months before the clinical symptoms of BSE became apparent. In addition, the 3 sources of BSE prion induced no detectable differences in the clinical signs, incubation periods, neuroanatomical location of vacuoles, or distribution and pattern of PrP(Sc) depositions in the brain.

The activation of P2X7 receptor induces cathepsin D-dependent production of a 20-kDa form of IL-1ß under acidic extracellular pH in LPS-primed microglial cells.

The potent pro-inflammatory cytokine, interleukin-1ß (IL-1ß), is synthesized as an inactive 33-kDa precursor (pro-IL-1ß) and is processed by caspase 1 into the bioactive 17-kDa mature form. The P2X7 receptor, an ATP-gated cation channel, plays an essential role in caspase 1 activation, production and release of mature bioactive 17-kDa form. We recently reported ATP induces the release of an unconventional 20-kDa form of IL-1ß (p20-IL-1ß) from lipopolysaccharide-primed microglial cells. Emerging evidence suggests physiological relevance for p20-IL-1ß; however, the underlying mechanisms for its production and release remain unknown. Here, we investigated the pathways involved in the ATP-induced production of p20-IL-1ß using lipopolysaccharide-primed mouse microglial cells. The activation of P2X7 receptor by ATP triggered p20-IL-1ß production under acidic extracellular conditions. ATP-induced p20-IL-1ß production was blocked by pepstatin A, a potent inhibitor of the lysosomal protease, cathepsin D. The removal of extracellular Ca(2+) inhibited the p20-IL-1ß production as well as ATP-induced cathepsin D release via lysosome exocytosis. The acidic extracellular pH also facilitated the dilatation of membrane pore after ATP stimulation. Since facilitation of pore dilatation results in cytolysis accompanied with cytoplasmic pro-IL-1ß leakage, our data suggest the leaked pro-IL-1ß is processed into p20-IL-1ß by cathepsin D released after ATP stimulation under acidic extracellular conditions.

Differential activities of plant polyphenols on the binding and internalization of cholera toxin in vero cells.

Plant polyphenols, RG-tannin, and applephenon had been reported to inhibit cholera toxin (CT) ADP-ribosyltransferase activity and CT-induced fluid accumulation in mouse ileal loops. A high molecular weight fraction of hop bract extract (HBT) also inhibited CT ADP-ribosyltransferase activity. We report here the effect of those polyphenols on the binding and entry of CT into Vero cells. Binding of CT to Vero cells or to ganglioside GM1, a CT receptor, was inhibited in a concentration-dependent manner by HBT and applephenon but not RG-tannin. These observations were confirmed by fluorescence microscopy using Cy3-labeled CT. Following toxin binding to cells, applephenon, HBT, and RG-tannin suppressed its internalization. HBT or applephenon precipitated CT, CTA, and CTB from solution, creating aggregates larger than 250 kDa. In contrast, RG-tannin precipitated CT poorly; it formed complexes with CT, CTA, or CTB, which were demonstrated with sucrose density gradient centrifugation and molecular weight exclusion filters. In agreement, CTA blocked the inhibition of CT internalization by RG-tannin. These data suggest that some plant polyphenols, similar to applephenon and HBT, bind CT, forming large aggregates in solution or, perhaps, on the cell surface and thereby suppress CT binding and internalization. In contrast, RG-tannin binding to CT did not interfere with its binding to Vero cells or GM1, but it did inhibit internalization.